Cultural eutrophication, the enrichment of aquatic systems with nitrogen and phosphorus caused by human activities, is linked to global increases in the frequency and intensity of harmful algal blooms (HABs) in fresh and marine waters (Glibert and others, 2005; Elser and others, 2007; Heisler and others, 2008; Glibert and Burford, 2017; Wurtsbaugh and others, 2019; Glibert, 2020). However, the relationship between nutrient enrichment and HABs is complex (Glibert and others, 2005; Glibert and Burford, 2017). For example, the concept of nitrogen and phosphorous limitation of phytoplankton assemblages in eutrophic-hypereutrophic systems where both nutrients are present at high concentrations is unclear. Algae have maximum cell quotas representing the internal concentration that saturates the cell’s capacity for further nutrient uptake and growth (Reynolds, 2006). If concentrations of nitrogen and phosphorous in the surrounding water simultaneously supersaturate algal growth requirements, neither nutrient should be limiting, regardless of which one is in lowest relative abundance (Reynolds, 1992, 1998, 1999; Davidson and others, 2012; Yang and others, 2020; Chorus and Spijkerman, 2021). Reynolds (1992, 1998) proposed saturation concentrations for dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP) of 0.10–0.13 and 0.03–0.10 milligrams per liter (mg/L), respectively (Chorus and Spijkerman, 2021). Under supersaturating concentrations of nitrogen and phosphorous, other factors such as light (low water clarity or self-shading during high biomass blooms), top-down control (grazing), or micronutrients (for example, silica, iron, manganese) limit algal growth and production (Reynolds, 2006).

Eutrophication, as well as the total maximum daily load and numerical nutrient criteria used for ecosystem management and restoration, is typically defined based on total nitrogen (TN) and total phosphorus (TP) rather than on specific nutrient forms (Organization for Economic Cooperation and Development, 1982; Dodds and others, 1998; Gibson and others, 2000; Florida Department of Environmental Protection [FDEP], 2012, 2020). Total nutrient measurements, however, include an assortment of different dissolved and particulate inorganic and organic forms (American Public Health Association and others, 2023) for which algae might have differential preferences or nutritional capabilities to exploit (Reynolds, 2006). These include DIN and SRP, the least energetically costly forms for autotrophic algae to assimilate (Reynolds, 2006). Although dissolved organic nutrient sources, such as urea, and more complex organic forms, such as amino acids, proteins, and humic substances, can be used by many algal taxa, more energy is required for cellular uptake and assimilation than would be needed for uptake and assimilation of inorganic forms (Glibert and Legrand, 2006). Furthermore, total nutrient measurements contain pools of potentially unavailable forms for most algae, including intracellular nutrients in the plankton themselves that are only available to heterotrophic algae able to ingest other microbes, as well as refractory particulate organic compounds and suspended-sediment-bound phosphorous (Reynolds, 2006). Understanding phytoplankton responses to specific nutrient sources rather than relying on measures of TN and TP is important when assessing nutrient limitation and eutrophication links to HABs (Ptacnik and others, 2010; Glibert and Burford, 2017).

The U.S. Geological Survey (USGS), in cooperation with the U.S. Army Corps of Engineers, Nova Southeastern University, and Florida Gulf Coast University, tested the concept of DIN and SRP limitation of phytoplankton assemblages in eutrophic-hypereutrophic systems with in situ nutrient enrichment mesocosms in the Caloosahatchee River, southwest Florida, where cyanobacterial HABs are a major concern (Rosen and others, 2017; Kramer and others, 2018; Metcalf and others, 2018; Lapointe and others, 2024; Krausfeldt and others, 2024; Badylak and others, 2025; Phlips and others, 2025). The short-term (72-hour incubation period) effects of ammonium (NH₄⁺), nitrate (NO₃⁻), and phosphate (PO₄³⁻) enrichment on phytoplankton dynamics were tested at three different times of the year corresponding to generalized algal growing seasons: pre-bloom (winter), bloom (spring/summer), and post-bloom (fall). We hypothesized that phytoplankton assemblages in the Caloosahatchee River will respond to pulses of excess inorganic nutrients over time scales ranging from hours to days with increased abundance and shifts in community structure. Particularly, we expected nitrogen to be limiting rather than phosphorous and that NH₄⁺ would cause a greater growth response than NO₃⁻. We also hypothesized that the effects of nutrient enrichment would differ across the three experiments due to variances in the natural phytoplankton assemblages present as inoculum and the meteorological and water-quality conditions during the season the mesocosms were conducted.

The Caloosahatchee River in southwest Florida flows westward from its headwater at Lake Okeechobee, the largest lake in the southeastern United States, into the Caloosahatchee estuary and the Gulf of America (fig. 1A). The mesocosm experiments were installed at the W.P. Franklin Lock and Dam (S-79, USGS station no. 02292900), the furthest downstream water control structure before the Caloosahatchee River discharges into the estuary (fig. 1B). The distance between S-79 and its connection to Lake Okeechobee at Moore Haven (S-77) is approximately 68 river kilometers; this stretch of the river from S-77 to S-79 is also called the C-43 canal (Taylor and others, 2022).

The Caloosahatchee River is an important environmental and economic resource for the State and local communities (South Florida Water Management District [SFWMD] and others, 2009). As a Class III waterbody, the river must meet water-quality standards that support its designated uses for recreation, fish consumption, and the “propagation and maintenance of a healthy, well-balanced population of fish and wildlife” (FDEP, 2020, p. 16). However, cyanobacterial HABs are a major problem in the river where noteworthy incidents occurred in 2016, 2018, 2021, and 2023 (Rosen and others, 2017; Kramer and others, 2018; Metcalf and others, 2018; Lapointe and others, 2024; Krausfeldt and others, 2024; Phlips and others, 2025). The Caloosahatchee River is classified as eutrophic to hypereutrophic based on TN, TP, chlorophyll-a (Chl-a), dissolved oxygen, and fecal indicator bacteria metrics (Bailey and others, 2009; Lapointe and others, 2018; Kahn and Betts, 2021; Taylor and others, 2022). Additionally, high concentrations of dissolved organic matter (DOM) color the river and reduce water clarity (fig. 1D; Liu and others, 2009; Chen and others, 2015; Badylak and others, 2025). To achieve water-quality restoration goals through nutrient total maximum daily load reductions, the FDEP implemented the Caloosahatchee Estuary Basin Management Action Plan in 2012 and published an updated version in 2020 (FDEP, 2020).

The Caloosahatchee River receives freshwater inflows and nutrient loading from Lake Okeechobee regulatory water releases and from local runoff and groundwater seepage from its watershed, the Caloosahatchee River Basin (Serna and others, 2020; Kahn and Betts 2021; Taylor and others, 2022). The Caloosahatchee River Basin has five subwatersheds. The W.P. Franklin Lock and Dam where the mesocosms were installed is at the boundary between the West Caloosahatchee and the Tidal Caloosahatchee subwatersheds and essentially demarcates the river from the estuary (fig. 1A; Taylor and others, 2022). Land use in the nontidal portions of the river basin (upstream of the W.P. Franklin Lock and Dam) is primarily citrus and sugar cane farms, natural areas, and improved pastures (SFWMD and others, 2009). Studies have shown that water quality and quantity in the Caloosahatchee River and its estuary are more strongly influenced by runoff from the tributaries within the Caloosahatchee River Basin than lake discharges, particularly during the wet season (Rumbold and Doering, 2020; Kahn and Betts, 2021; Taylor and others, 2022; Montefiore and others, 2024). However, others, have demonstrated that lake discharges have important effects on downstream phytoplankton dynamics and that cyanobacterial HABs might be seeded from Lake Okeechobee rather than originating in the river (Phlips and others, 2023, 2025; Urakawa and others, 2023; Badylak and others, 2025).

In situ nutrient enrichment mesocosms were conducted to test phytoplankton responses to NO₃⁻, NH₄⁺, and PO₄³⁻ enrichment above background levels to better understand how pulsed nutrient loading influences phytoplankton dynamics in the hypereutrophic and colored Caloosahatchee River. During all three deployments, the concentrations of NO₃⁻, NH₄⁺, and PO₄³⁻ added to the mesocosm tanks declined within 24 hours of dosing, but this was not always accompanied by increases in phytoplankton abundance metrics. The effects of nutrient enrichment varied across the three seasons during which the experiments were conducted—summer (June), fall (September), and winter (February). Phytoplankton abundance metrics were significantly elevated in the A treatments after 72 hours of exposure in the June 2020 and February 2021 deployments, whereas N treatments had a significant effect only in June 2020, supporting the hypothesis that NH₄⁺ has a stronger influence on phytoplankton dynamics than NO₃⁻. Furthermore, the hypothesis that nitrogen rather than phosphorus would be limiting was supported by the absence of significant responses to P treatments during any deployment. Despite increased abundance with A and N treatments, no significant shifts in phytoplankton assemblage structure were observed during the 72-hour experiments. Growth-rate measurements confirmed that only taxa which were already dominant at the start of the experiments exhibited increased growth rates that were driving elevated total abundance. Furthermore, the results of this study demonstrated that Microcystis, the dominant HAB-former in Lake Okeechobee (Paerl and others, 2020; Lapointe and others, 2024; Krausfeldt and others, 2025), maintained relatively low baseline cell concentrations in the Caloosahatchee River at the time of these experiments and that Microcystis population growth was not stimulated by NO₃⁻, NH₄⁺, or PO₄³⁻ enrichment during the 72-hour exposure period. The strongest effect on phytoplankton dynamics in all mesocosms was due to enclosure time, with significant shifts in community structure and increased abundance from T0 to T72 in all treatments, including the controls, during all three deployments. This suggests that nutrients are not the primary limiting resource for phytoplankton in the Caloosahatchee River. We hypothesize that an enclosure effect improved water clarity in the tanks and potentially released phytoplankton from light limitation. The Caloosahatchee River has high levels of DOM which color the river causing low water clarity and potentially light-limiting conditions for phytoplankton (Liu and others, 2009; Chen and others, 2015).

Mean DIN and SRP concentrations in the Caloosahatchee River at water-quality monitoring site S-79 during 2015–21 were above the saturation thresholds proposed by Reynolds (0.10–0.13 mg/L for DIN and 0.03–0.10 mg/L for SRP), suggesting that phytoplankton in the river should not be limited by either nutrient. Reynolds (1992, 1998, 1999) stated that above these saturation thresholds, limitation of algal growth by either nutrient is highly improbable and the ratio of nitrogen to phosphorus is irrelevant because the cell reaches its maximum quota for both nitrogen and phosphorus in the optimal ratio before either becomes depleted enough in the water to be considered limiting (Chorus and Spijkerman, 2021). Cells have limits on how fast they can take up and assimilate nutrients, as described by the Michaelis-Menten, Droop, and Monod equations (Reynolds, 2006; Dodds and Whiles, 2020), and it follows that above saturating concentrations, additional nutrients will not be taken up if maximum cell quotas and optimal stoichiometry have been met. However, NH₄⁺, NO₃⁻, and PO₄³⁻ added to the mesocosms were significantly drawn out of solution within 24 hours after each dosing application even though baseline (T0) concentrations of DIN (NO₃⁻+NO₂⁻+ NH₄⁺) and SRP were above the Reynolds’ saturation thresholds during all three experiments, indicating that the microbial assemblages were not saturated in their uptake potential for DIN or SRP. Luxury uptake and storage of nutrients by phytoplankton might explain why removal of added nutrients from the water column did not always translate to increased phytoplankton abundance, such as in the September 2020 deployment and with P treatments during any deployment (Kromkamp, 1987; Reynolds, 2006; Flores and others, 2019; Solovchenko and others, 2019). Uptake by bacterioplankton and sorption to abiotic particles can also deplete inorganic DIN and SRP (Kirchman, 1994; Hampel and others, 2020; Sörenson and others, 2020). Nevertheless, significant increases in phytoplankton abundance in the A and N treatments demonstrate periodic nitrogen limitation in the Caloosahatchee River despite its eutrophic status and inorganic nutrient concentrations exceeding Reynolds’ saturation thresholds (Paerl and others, 2020). Though our study is the first nutrient enrichment experiment conducted in the Caloosahatchee River, previous nutrient enrichment studies in Lake Okeechobee and the Saint Lucie River, the lake’s eastward riverine outflow, also documented nitrogen limitation in mesocosms incubated over 24 hours (Kramer and others, 2018) or 72 hours (Havens and others, 1996; Havens and East, 1997; Kramer and others, 2018). Similarly, Ma and others (2015) reported nitrogen rather than phosphorus limitation of phytoplankton from mesocosm experiments conducted in Lake Taihu, China, a hypereutrophic lake comparable to Lake Okeechobee; however, significant biomass increases in nitrogen versus control treatments were not evident until day 12 of the incubation in the Lake Taihu study, whereas we observed significant increases starting at T72 in two of our three deployments.

Light limitation in the highly colored, DOM-rich Caloosahatchee River is a possible explanation for the observed nitrogen limitation despite background concentrations of DIN above saturation concentrations because light-limited cells are less able to use available nutrients. Without adequate light for photosynthesis, cells cannot produce sufficient glucose to fuel cellular respiration and produce enough ATP to power the transport of nutrients across the cell membrane, an active process requiring energy (Falkowski and Raven, 2007). Light limitation has been documented as another driving factor of phytoplankton dynamics in Lake Okeechobee (Philips and others, 1997; Havens and others, 2003) but has not been tested explicitly in the Caloosahatchee River and was outside the scope of our experiments. However, enclosure in the mesocosm tanks seems to have improved water clarity, as inferred by reductions in fDOM and turbidity over time, potentially releasing phytoplankton from light limitation and allowing them to use available background nutrients for growth, resulting in the significant increases in phytoplankton abundance in controls and all treatment tanks during the 72-hour period. The stronger influence of enclosure time than nutrient treatments on phytoplankton growth in this study suggests that nutrients are not the primary limiting resource for phytoplankton in the Caloosahatchee River. Flocculation and sedimentation of fDOM, suspended sediments and other turbidity causing particles is a potential mechanism for improved water clarity in the tanks along with potentially higher incident photosynthetically active radiation on algal cells from light quanta entering the tanks from above and being reflected off the side and bottom walls of the plexiglass tanks. Significantly greater phytoplankton abundance in the A and N treatments at T72 relative to T72 controls indicates that additional nitrogen inputs, particularly NH₄⁺, supports growth beyond what is achieved using ambient nitrogen once the primary limiting resource has been supplied. However, even with the added nitrogen, phytoplankton abundance in the A and N treatments did not increase enough to result in a visible accumulation of biomass that could be defined as a bloom. Observations from this study allude to the potential effects of water clarity on phytoplankton response to inorganic nutrient loading in the Caloosahatchee River.

Consistent with other phytoplankton studies in the Lake Okeechobee system, cyanobacteria dominated the phytoplankton assemblages during all three deployments (McPherson and La Rose, 1982; Havens and others, 1996, 2003; Philips and others, 1997). The hypothesis that the relative abundance of cyanobacteria, compared to other algal groups, would shift with nutrient amendments and that diazotrophic cyanobacteria would be favored when nitrogen:phosphorous ratios declined (P treatments), was not supported. Havens and East (1997) also did not observe significant shifts in the relative abundance of cyanobacteria compared to other algal groups with nutrient amendments, possibly because the systems were already dominated by cyanobacteria, the group that would be expected to outcompete others under warm, eutrophic, low water-clarity conditions such as those in the Lake Okeechobee system (Paerl and others, 2001). Shifts in cyanobacterial assemblages were not observed either. The non-heterocystous, colonial genera Aphanocapsa and Merismopedia were always the dominant cyanobacteria in our study. Both genera belong to the family Microcystaceae in the order Chroococcales. Although Aphanocapsa and Merismopedia are not as well known for causing freshwater HABs compared to other species such as Microcystis, studies have documented that these species can form toxic HABs (Jakubowska and Szelag-Wasielewska, 2015; de J. Magalhães and others, 2019; Keliri and others, 2021). Kramer and others (2018) reported abundant Aphanocapsa during the state of emergency Microcystis bloom of 2018 in Lake Okeechobee and the Saint Lucie River. Microcystis, also a non-heterocystous, colonial genus in the family Microcystaceae, has been reported as the dominant taxa during blooms in Lake Okeechobee (Paerl and others, 2020; Lapointe and others, 2024; Krausfeldt and others, 2025). In our study, Microcystis exhibited relatively low baseline cell concentrations, and the NH₄⁺, NO₃⁻, or PO₄³⁻ enrichments did not stimulate a burst in population growth within a 72-hour period. Microcystis failure to respond to experimental nutrient additions could simply indicate that more than 72 hours is needed to trigger a Microcystis bloom or that it is limited by a resource other than nitrogen or phosphorus. The drivers or processes that enable Microcystis to gain a competitive advantage resulting in a population explosion from relatively low baseline concentrations to a HAB formation remain poorly understood (Wilhelm and others, 2020). The results of our experiments contribute to our knowledge of Microcystis ecology and bloom dynamics in the Caloosahatchee River that may be relevant to HAB management strategies.

In conclusion, the results of our study indicate that phytoplankton in the Caloosahatchee River is co-limited by nitrogen and a second unknown factor, which we hypothesize is light in this colored, DOM-rich system. The NH₄⁺, NO₃⁻, and PO₄³⁻ added to enclosed Caloosahatchee River water were rapidly removed from solution, but the fate of those nutrients within algal cells (such as use for growth and replication or kept in storage compounds), their relative uptake by different algal taxa or compartments (bacterioplankton or sorption to sediments), and their role in triggering a HAB event are still not well understood. Longer experiments could be useful to capture population and assemblage responses along the full range of the algal growth curve from the lag phase through the exponential phase, the stationary phase, and senescence. This could allow identification of nutrient threshold levels leading to bloom initiation, lag times between initial exposure and response, and assemblage successional patterns which can support effective management and prediction of HABs. This study offers insights into the relationship between inorganic nutrient loading and phytoplankton dynamics in the Caloosahatchee River, which had not been previously tested, and revealed the potential role of water clarity in hindering HAB formation despite supersaturating nutrient levels. The study results could be used to evaluate future strategies for controlling cyanobacterial HABs in the Caloosahatchee River, where it remains unclear if HAB events are independent of HABs in its headwater, Lake Okeechobee, or whether they are transported to the river through necessary regulatory water releases.